A protein analogous to profilin previously isolated by others from human platelets and calf spleen has been purified from Acanthamoeba castellanii. The protein consists of a single polypeptide chain of molecular weight about 12,000-14,000. It probably forms a 1:1 molar complex with monomeric actin and inhibits the rate of polymerization of actin by inhibiting the first step in the polymerization process - the nucleation reaction in which approximately 3 actin monomers interact to form a nucleus that can be elongated by addition of other actin subunits. Profilin does not seem to inhibit the elongation steps nor the final extent of polymerization. Acanthamoeba profilin has been shown to be present in sufficient quantities in the cell to account for all of the non-polymerized actin, about 50% of the total actin in the cell. Acanthamoeba profilin has a pI of about 6.4 and contains about 28% alpha-helix and 29% beta-confirmation. This is the first isolation of profilin from a non-mammalian source. The isolation procedure (40% recovery) should be applicable to the isolation of similar proteins from other sources. Profilin is probably one important elment in the process by which the time and place of actin polymerization in the cell is regulated.